The experiments outlined in this proposal are aimed at identifying and controlling the ensemble of conformations that comprise the native state of a protein. In order to appreciate how a protein's sequence encodes a folded, functional molecule, we need to understand much more than just its native structure. Encoded in the primary sequence is the entire energy landscape. Non-native regions of the landscape are important for directing the stability and folding of a protein, and modulations in this ensemble play a crucial role in misfolding, signaling, allostery, and turnover. A major hurdle in our ability to go from sequence to function is our lack of understanding of these non-native states. Many different sequences can encode the same three dimensional structure, yet their function and dynamics vary dramatically; small variations can have effects that range from undetectable to pathological. These differences are often a consequence of subtle changes in non-native regions of the landscape. Here, we propose experiments directed at determining and controlling different features of the landscape using a mix of new and established experimental techniques and systems. Specifically, the aims of this proposal are: Specific aim 1: Single molecule studies of protein conformational changes a. Explore the unusual mechanical properties of non-native states b. Obtain energetic information from these inherently non-equilibrium studies c. Determine the mechanical coupling in repeat proteins Specific aim 2: Examine perturbations of the free energy landscape. a. Explore the energy landscape of thermophiles b. Probe the energy landscape of RNase H using mutagenesis and MPAX:mis-incorporation proton alkyl exchange Specific Aim 3: The de novo design of function through modulation of conformational ensembles. a. Design of a controllable allosteric switch b. Demonstrate control by ligand binding and mechanical tension [unreadable] [unreadable] [unreadable]